Process for stabilizing the content of glycated protein of a sample on a matrix material

ABSTRACT

The invention concerns a method for stabilizing the content of glycated protein in a sample on a matrix material, which is characterized in that the matrix material is impregnated with boric acid buffer with a pH which is larger or equal to 10.5 or a transition metal salt as well as an appropriate matrix material, an element for collecting, transporting and storing sample material to be analyzed with such a matrix material and a system containing such an element and a sealable covering.

This is a continuation of application No. 08/702,972, filed Aug. 26,1996.

BACKGROUND OF INVENTION

The invention concerns a process for stabilizing the content of glycatedprotein of a sample on a matrix material and an appropriately treatedmatrix material for this. In addition the invention concerns an elementfor collecting, transporting and storing sample material to be analysedcontaining an absorptive matrix material and a system containing such anelement and a sealable covering in which the element can be transported.

The glycation of haemoglobin and serum proteins is increased in patientswith diabetes mellitus. The increase is dependent on the glucoseconcentration and the incubation period of protein with glucose. Inthese cases the serum proteins, including haemoglobin, are not glycatedenzymatically but rather by means of an uncatalysed chemical reaction ofglucose with amino groups of proteins. Experts assume that theconcentration of a particular protein-glucose adduct reflects theglucose concentration over a particular period as well as the turn-overrate of the protein. Glycated haemoglobin is regarded as an indicator ofthe average blood glucose concentration during the last two to threemonths before the blood collection and examination. Glycated serumprotein shows the blood glucose concentration during a shorter period oftime. The determination of glycated protein such as glycated haemoglobin(HbA_(1c)) or glycated serum protein is therefore considerably importantfor the long-term glycemic control of diabetes patients.

In order to examine blood for the content of glycated protein the samplemust often be transported to a far distant laboratory. The content ofglycated protein in the sample should not change during this transportperiod and during a possible subsequent waiting period. The examinationof blood samples which had been stored for a long period for glycatedhaemoglobin is reported in Clinical Chemistry 29, 1080-1082 (1983). Thisshows that whole blood can be stored up to 21 days at room temperaturewith essentially no change in the HbA_(1c) content.

However, the transport of liquid blood samples is complicated andinvolves risks such as breakage of the transport vessel. In addition thepuncture of a vein is necessary to collect whole blood although theamounts obtained by withdrawing capillary blood from the finger padwould be sufficient for the analysis. Thus methods have been developedfor the transport and analysis of smaller amounts of blood in whichcapillary blood is applied to filter paper and allowed to dry there. Thefilter paper is subsequently transported to the site of the examination.Here a disk containing the sample is cut out from the filter paper,eluted and the eluate is examined. The report in Clinical Chemistry 28,386-387 (1982) refers to such a method. In this report it is stated thatthe content of glycated protein changes considerably compared to theoriginal sample during blood sample storage on filter paper. Afterstorage of whole blood on filter paper considerably increased measuredvalues for glycated protein are found.

The impregnation of filter paper with glucose oxidase to prevent theincrease in the content of glycated haemoglobin caused by storage ofblood on filter paper is described in Clinical Chemistry 32, 869-871(1986). However, impregnation with glucose oxidase was not able tocompletely prevent the increase of glycated protein. The false increasein the values can only be reduced by this measure. A furtherdisadvantage of impregnating with glucose oxidase is its own instabilityduring storage under the usual storage conditions. Similar conclusionsare reached by an article in Diabetes Care 10, 352-355 (1987). Here itis reported that the treatment of filter paper with glucose oxidase orwith ethanol cannot satisfactorily prevent a false increase in thevalues for glycated haemoglobin when blood is stored on filter paper.

OBJECTS AND SUMMARY OF THE INVENTION

The object of the present invention was therefore to stabilize thecontent of glycated protein in a sample when stored on a matrixmaterial. After storage of the glycated protein on a matrix material avalue should be found for the glycated protein which corresponds to thatfound after sample collection and before storage.

This object is achieved by the subject matter characterized in moredetail in the patent claims.

BRIEF DESCRIPTION OF THE DRAWING

The invention concerns a process for stabilizing the content of glycatedprotein of a sample on a matrix material by impregnating the matrixmaterial with a boric acid buffer with a pH greater than or equal to10.5. A comparable stabilization is also possible when the matrixmaterial carries a transition metal salt.

The invention in addition concerns the matrix material for taking up thesample material which is to be examined for its content of glycatedprotein which is characterized in that it is impregnated with boric acidbuffer with a pH larger than or equal to 10.5 or it carries a transitionmetal salt.

An additional subject matter of the invention is an element forcollecting, transporting and storing sample material to be analysedcontaining an absorptive matrix material wherein the element ischaracterized in that the matrix material is impregnated with boric acidbuffer with a pH larger than or equal to 10.5 or it carries a transitionmetal salt.

Finally a subject matter of the invention is a system containing anelement characterized as above and a sealable covering in which theelement can be transported which is characterized in that the element issuch as has already been characterized above as being according to theinvention.

Within the scope of the present invention matrix materials denoteabsorptive materials which are capable of absorbing a liquid containingglycated protein. Glycated protein, i.e. protein carrying sugarresidues, is mainly located in the blood but also in sample materialsderived from blood such as serum or plasma and moreover also in otherbody fluids such as urine or saliva. Materials which can absorb suchsample materials are preferably fibres but can also be in principlenon-fibrous. Preferred fibrous absorptive matrix materials are fleeces,fabrics or knitted fabrics. Fleeces are quite especially preferred. Thefibrous matrix materials can contain glass, cellulose, polyester fibresand also viscose and polyvinyl alcohol. Fleece materials containingmeltable copolyester fibres in addition to glass fibres, polyesterfibres, polyamide fibres, cellulose fibres or cellulose derivativefibres as described in the European Patent Application 0 571 941 canalso be used advantageously as the matrix material. Non-fibrousmaterials can for example be membranes.

According to the invention it has turned out that sample materialcontaining glycated protein that is located on a matrix material can bestored very well without any essential change in the content of glycatedprotein if the matrix material is impregnated with boric acid bufferwith a pH of greater than or equal to 10.5 or if the matrix materialcarries a transition metal salt. In this case the concentration of theboric acid buffer is of secondary importance. Particularly good resultsare obtained if the boric acid buffer has a pH value of more than orequal to 11. Suitable buffer concentrations are in the range between 300and 1000 mmol/l, which corresponds to about 18.6-62 g/100 ml.

Transition metal salts have a similarly good stabilizing action such asnickel or copper salts. Nickel salts are particularly preferred.Water-soluble transition metal salts are preferably used according tothe invention. Corresponding chlorides are for example well suited. Inorder to be active according to the invention transition metal saltconcentrations on the matrix material of more than 5 g/m² andparticularly preferably of more than 10 g/m² have proven to be suitableaccording to the invention.

Sample material containing glycated protein such as for example glycatedhaemoglobin which has been applied to a matrix material as describedabove has resulted in values for glycated protein that are comparablewith the original concentration even when measured after a long periodof storage at increased temperature. All liquids come into considerationas the sample material which have already been mentioned above. Blood orsamples derived from blood such as plasma or serum are quite especiallypreferred. However, according to the invention in principle all liquidscan be used that can contain glycated proteins.

In addition all proteins come basically into consideration as glycatedproteins which are formed by chemical reaction of a protein withglucose. Glycated haemoglobin which is also named HbA_(1c) is ofparticular importance. Glycated albumin or glycated serum proteinsshould also be mentioned in this connection.

A matrix material as described above which can be used according to theinvention to stabilize the content of glycated protein of a sample canalso be contained in an element for collecting, transporting and storingsample material to be analysed. Such an element can for example be madelike the HbA_(1c) Via Post® sold by Boehringer Mannheim. This element isdescribed in Klin. Lab. 39, 1080-1082 (1993). Instead of a roundunimpregnated section of filter paper which is attached to a carriermaterial, a treated matrix material according to the invention asdescribed above can be used onto which the liquid sample material and inparticular blood can be applied in such a way that it is absorbed by thematrix material. In this way the element can be transported to theexamination site where the sample is eluted from the matrix material andthe eluate can be examined. By using the matrix material according tothe invention the sample material contained therein can be stored for along period without major changes in the content of glycated protein.The application of the matrix material according to the invention hasproven to be particularly suitable in particular for the interestingparameter HbA_(1c).

An element has proven to be particularly suitable for application of thepresent invention which contains a first and a second layer ofabsorptive matrix material which are arranged next to and touching oneanother on an inert carrier material in a contact that enables transferof liquid in such a way that liquid can pass from the first into thesecond layer when the first layer is filled with liquid and the firstlayer can be completely separated from the second layer afterapplication and drying of the sample material. Such an element isdescribed in the German Patent Application P 19 523 061.2. According tothe invention a matrix material is used for the first layer which isimpregnated as described above and either carries a boric acid bufferwith a pH larger than or equal to 10.5 or a transition metal salt.

An important property of the absorptive matrix materials that can beused in such an element is their absorptivity. The absorptivity of thematrix material of the first layer should be equal to or larger thanthat of the neighbouring layer. This avoids interfering suction effectsfrom developing when sample material is applied to the first layer.

The absorptivity can be determined according to DIN 53106. For thispurpose the lower end of samples of 200+/−1 mm in length and 15+/−0.1 mmin width are immersed perpendicularly 25 mm into distilled water and thedistance which the water migrates within 10 min is measured in mm. Aperson skilled in the art knows how different absorptivities can beadjusted in matrix materials with the same components. For example whenmanufacturing fleeces different thicknesses can be used. The thicker thefibres used the lower is the absorptivity. A further method is to varythe density of fleeces. The absorptivity is reduced by an increase indensity.

When using fabrics, fabrics with finer fibres have a higher absorptivitythan fabrics with coarser fibres. However, the absorptivity can also becontrolled by different types of twisting of the threads. In additionvariations in the absorptivity can be achieved via the type of weaving.Further possibilities for varying the absorptivity can be achieved byusing different mixtures of fibres. Thus for example the absorptivity isreduced by the addition of hydrophobic fibres.

Stiff materials come into particular consideration as the inert supportmaterial on which the matrix material layers are located such as forexample plastic foil, cardboard, coated paper etc. The matrix materiallayers are attached to the inert support material in such a way that theuptake of liquid by the matrix materials is not impaired. This can beachieved by using a double-sided adhesive tape or for example also byusing hot-melt adhesive.

The matrix material layers must be attached to the inert supportmaterial in such a way that the first layer can be completely separatedfrom the second layer after applying and drying the liquid samplematerial. This is then in particular possible when the first layer isattached only relatively loosely or at certain points only.

The two matrix material layers must be located on the support materialnext to and touching one another in such a way that liquid can pass fromthe first layer into the second layer when the first layer is filledwith liquid. This is then possible when at least the edges of the twolayers are touching. It is even better, however, if there is a slightoverlap of the two layers. It is particularly preferred that the layersare arranged such that the second layer slightly overlaps the firstlayer.

The size of the matrix material layers must be selected such that thefirst layer, which is later also to be used as the analytical layer, canbe completely filled with the sample liquid. Excess sample liquid isthen taken up by the second layer. The amount of sample which isadequate to determine a particular analyte depends on the type ofanalyte to be determined. However, as a rule 5-20 μl and usually 10 μlsample is adequate. This volume must be taken up by the first matrixlayer and capable of being eluted again later. For safety reasons thesecond matrix layer which has the function of a suction layer should beable to absorb a larger volume. Suction volumes of 10-50 μl, preferably10-30 μl particularly preferably 20 μl are usually adequate for thispurpose. It is expedient that the usual dimensions of the matrixmaterial layers are such that the suction volume of the two matrixmaterial layers taken together is at least 30 μl and preferably at least50 μl. Such a dimension ensures that the same amount of sample isapplied on the first matrix layer of various elements according to theinvention with small as well as with large drops of liquid. In order toachieve an adequate suction volume the smaller first layer usually hasan area of 3×3 to 8×8 mm.

The arrangement of matrix material layers described above enables ahomogeneous distribution of liquid sample material to be achieved in thefirst layer. Due to the fact that the first layer is completely filledwith liquid sample material, reproducible amounts always reach theanalysis after separation and elution of the first layer.

In order to transport an element according to the invention afterapplying the sample material to the analytical station it has alsoproven to be expedient to transport it in a sealable covering. Thecovering and element thus form a system. An envelope is for examplesuitable as the sealable covering such as a letter envelope whichencompasses a front part and two side parts as well as a backflap and asealing flap with which the envelope can for example be glued. Such acovering is also described in the German Patent Application P 19 523061.2.

The invention is elucidated further in the following examples.

EXAMPLE 1 Stabilizing HbA_(1c) by Boric Acid Buffer

A first layer (1) of an absorptive matrix material is fixed with the aidof a double-sided adhesive tape (4) to a polyester support foil (3) ofdimensions 49×6 mm with a semicircular punch hole (5) of 5 mm at itsshort-sided end as shown in FIG. 1 in such a way that 0.5 to 1 mm of itswidth is glued onto the adhesive tape (4). The later detachability ispositively influenced by this relatively narrow attachment. The secondlayer (2) of the absorptive matrix material is glued in a width of 5 mmor more.

A fleece which has been manufactured on a paper machine which has thefollowing data is used for the first layer of absorptive matrixmaterial:

80 parts polyester fibres (fibre diameter 1.7 Dtex), 20 parts viscose,20 parts polyvinyl alcohol; area weight 80 g/m²; suction height 102 mm(DIN 53106).

This fleece was impregnated with one of the boric acid buffers listed intable 1 (62 g boric acid suspended in 800 ml distilled water, adjustedto the desired pH value with 5 mol/l potassium hydroxide solution andfilled up to 1000 ml with distilled water), dried at 50° C. andsubsequently cut to a size of 6×6 mm. This matrix takes up ca. 10 μl ofliquid.

A fleece is used for the second layer of absorptive matrix materialwhich corresponds to the first layer but is not impregnated with boricacid buffer.

Ca. 10 μl EDTA blood containing 5.1% HbA_(1c) supplemented with 500mg/dl glucose is applied in each case to the elements according to theinvention manufactured in this manner and dried at room temperature forat least 2 hours. In order to simulate a transport the dried samplecarriers were stressed for 5 days at 35° C. in shipping envelopes.

After removing the first matrix layer with tweezers, the matrix materialis eluted in 1 ml haemolysis reagent for the Tina-quant® test ofBoehringer Mannheim GmbH (Germany) (order number 1 488 457).Subsequently HbA_(1c) is determined according to the immunologicalmethod of determination of Boehringer Mannheim GmbH (Germany) on aHitachi 717 instrument from Boehringer Mannheim GmbH using reagent withorder number 1 488 414 from Boehringer Mannheim GmbH.

The measured results are summarized in table 1 for elements in which thematrix material contains no boric acid buffer or boric acid buffer ofvarious pH values.

TABLE 1 Storage conditions 5 days at 2 to 8° C. 35° C. Matriximpregnated with HbA_(1c) (%) HbA_(1c) (%) without boric acid 5.6 11.2boric acid pH 6.3 5.3 10.6 boric acid pH 7.0 5.4 10.1 boric acid pH 8.05.3 9.1 boric acid pH 9.0 5.2 7.9 boric acid pH 10.0 5.5 7.0 boric acidpH 10.5 5.0 6.0 boric acid pH 11.0 5.2 5.5 boric acid pH 12.0 5.1 5.3

The result shows that boric acid buffer above a pH value of 10.5 leadsto a stabilization of the non-enzymatically glycosylated protein to suchan extent that after temperature stress adequate unchanged concentrationvalues are present.

EXAMPLE 2 Stabilization of HbA_(1c) by Nickel(II)Chloride

Analogously to example 1 elements for collecting, transporting andstoring sample material to be analysed are manufactured in which,however, the first matrix layer has been impregnated with a NiCl₂concentration series between 0 and 200 mmol/l NiCl₂ (0; 9.6; 14.4; 19.2;24.0 and 48 g/l nickel chloride-6 hydrate dissolved in 1000 ml distilledwater) in such a way that various nickel salt concentrations are presentin the matrix as listed in table 2. A blood sample such as the one usedin example 1 containing 5.1% HbA_(1c) supplemented with 500 mg/dlglucose was used. Determination of HbA_(1c) as in example 1 resulted inthe concentration values listed in table 2.

TABLE 2 Storage conditions 5 days at Matrix impregnated with 2 to 8° C.35° C. NiCl₂ (g/m²) HbA_(1c) (%) HbA_(1c) (%) 0 5.6 11.2 4.2 5.4 8.2 6.35.1 6.3 8.4 5.2 5.7 10.5 4.9 5.8 21.0 5.3 4.9

Having described the preferred embodiments of the invention with respectto the accompanying drawing, it is to be understood that the inventionis not limited to these precise embodiments and that various changes andmodifications may be effected therein by one skilled in the art withoutdepartment from the scope and spirit of the invention as defined in theappended claims.

What is claimed is:
 1. A matrix material comprising a fibrous material which has been impregnated with a boric acid buffer at a pH of at least 10.5, or which carries a transition metal salt and which has a glycated protein absorbed thereon.
 2. The matrix material of claim 1, wherein said water soluble transition metal salt is a nickel salt or a copper salt.
 3. The matrix material of claim 2, wherein said water soluble transition metal salt is a nickel salt.
 4. The matrix material of claim 2, wherein said water soluble transition metal salt is a copper salt.
 5. A dried matrix material having impregnated thereon a water soluble transition metal salt and a glycated protein.
 6. The matrix material of claim 5, wherein said water soluble transition metal salt is a nickel salt or a copper salt.
 7. The matrix material of claim 6, wherein said water soluble transition metal salt is a nickel salt.
 8. The matrix material of claim 6, wherein said water soluble transition metal salt is a copper salt. 